CN106701864B - Method for improving validamycin yield - Google Patents
Method for improving validamycin yield Download PDFInfo
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- CN106701864B CN106701864B CN201510409235.0A CN201510409235A CN106701864B CN 106701864 B CN106701864 B CN 106701864B CN 201510409235 A CN201510409235 A CN 201510409235A CN 106701864 B CN106701864 B CN 106701864B
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Abstract
The invention relates to the field of microorganisms, and particularly provides a method for improving the yield of validamycin. The validamycin is a plant-derived bactericide, is a water-soluble antibiotic-glucoside compound produced by streptomyces hygroscopicus validamycin variants, and has A, B, C, D, E, F components, and the main active substance is validamycin A. The invention improves the yield of validamycin by further optimizing the specific components of the streptomyces hygroscopicus validamycin variant fermentation medium and other culture conditions. The yield of the validamycin A can reach 27000 mu g/mL, and the validamycin A is simple in scheme, low in cost and suitable for industrial large-scale production.
Description
Technical Field
The invention relates to the field of microorganisms, and particularly provides a method for preparing validamycin by fermenting streptomyces hygroscopicus validamycin A varieties.
Background
Validamycin (jingangmycin) is a plant-derived bactericide, is a water-soluble antibiotic-glucoside compound produced by streptomyces hygroscopicus validamycin variants, and has A, B, C, D, E, F components in total, wherein the main active substance is validamycin A, and the content of the component A in general products indicates the specification and the quality of the products. Validamycin is an antibiotic produced by actinomycetes, has strong systemic property, is easily absorbed by bacterial cells and quickly conducts in the bacterial cells, and interferes and inhibits the growth and development of the bacterial cells. The validamycin is mainly used for wheat sheath blight, can also be used for rice false smut, corn big and small leaf spot, crop diseases of vegetables, cotton, beans and the like, and has the advantages of high control effect, no phytotoxicity, no pollution and the like.
At present, the method for preparing validamycin in the prior art has the problems of low yield and large raw material consumption.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for producing validamycin by fermenting streptomyces hygroscopicus validamycin A. The streptomyces strain of the present invention is a conventional streptomyces strain, and can be obtained commercially or isolated from rice by a method conventionally used by those skilled in the art. The recombinant strain can also be obtained by a method disclosed in literature validamycin strain natural breeding and prolificacy research (Nature science and sciences journal of the university of Hunan province 2009,32(2) Author Shihua, Qifengpei, Van Guang and Xuancai (chemical and chemical industry institute of the university of Hunan province, China Changsha 410081)).
The invention provides a method for producing validamycin by fermenting streptomyces hygroscopicus validamycin variants, which specifically comprises the following steps:
step (1): inoculating the streptomyces hygroscopicus validus var validus into a slant strain culture medium, culturing until spores are mature, and preparing a seed solution for later use;
step (2): inoculating the seed solution into a fermentation culture medium containing 0.001-0.005 g/L of cellulase for fermentation culture to obtain a fermentation liquid;
and (3): extracting validamycin from the fermentation liquor.
The strain culture medium in the step (1) comprises the following components: 25-30 g/L of corn flour, 20-22 g/L of soybean cake powder and 10-15 g/L, NaCl 1-3 g/L, KH of yeast powder2PO40.5～1g/L，CaCO30.1-0.2 g/L, and the balance of water; adjusting the pH value to 6.8-7.2, preferably 7.0.
Further, the culture medium components in step (1) of the invention are preferably: 30g/L corn flour, 22g/L soybean cake powder and 10g/L, NaCl2g/L, KH g/yeast powder2PO40.8g/L，CaCO30.2g/L and the balance of water.
The fermentation medium in the step (2) of the invention also comprises the following components: 90-100 g/L corn flour, 25-30 g/L soybean cake powder and 5-8 g/L, NaCl 1-2 g/L, KH yeast powder2PO41～3g/L、CaCO30.4-0.6 g/L of water; adjusting the pH value to 7.0-7.5, preferably 7.2.
The cellulase added into the fermentation medium can generate good synergistic effect with other medium components, and the yield of validamycin is effectively improved. The addition of the cellulase can decompose a part of cellulose in the raw material to generate glucose, thereby being beneficial to the absorption of thalli.
Further, the fermentation medium component described in step (2) of the present invention preferably further comprises: 100g/L corn flour, 25g/L soybean cake powder, 5g/L, NaCl1g/L, KH g yeast powder2PO41.5g/L，CaCO30.5g/L and water.
The water is distilled water or deionized water. Distilled or deionized water can avoid the entrainment of impurity ions.
The fermentation culture in the step (2) is shaking culture, the rotation speed of the shaking culture is 200-250 r/min, the fermentation temperature is 35-45 ℃, and the fermentation time is 90-110 h.
As an overall technical solution, the present invention provides a more targeted solution, specifically as follows:
the invention provides a method for producing validamycin by fermenting streptomyces hygroscopicus validamycin variants, which comprises the following steps:
step (1): inoculating the streptomyces hygroscopicus validus var validus into a slant strain culture medium, culturing until spores are mature, and preparing a seed solution; the strain culture medium comprises the following components: 30g/L corn flour, 22g/L soybean cake powder and 10g/L, NaCl2 g/2 g/L, KH yeast powder2PO40.8g/L、CaCO30.2g/L and the balance of water, and adjusting the pH value to 7.2; standby;
step (2): inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid; the fermentation medium comprises the following components: 100g/L corn flour, 25g/L soybean cake powder, 5g/L, NaCl1g/L, KH g yeast powder2PO41.5g/L，CaCO30.5g/L of cellulase, 0.003g/L of cellulase and the balance of water; adjusting the pH to 7.2;
and (3): extracting validamycin from the fermentation liquor.
A fermentation medium comprising the following components: 90-100 g/L corn flour, 25-30 g/L soybean cake powder and 5-8 g/L, NaCl 1-2 g/L, KH yeast powder2PO41～3g/L、CaCO30.4-0.6 g/L, 0.001-0.005 g/L cellulase, and the balance water. The fermentation medium provided by the invention has good application in validamycin production.
The validamycin product prepared by the method has the advantages that the yield of validamycin production can be obviously improved by adding the cellulase in the fermentation culture medium in a reasonable amount, the method is simple, and the cost is low. Therefore, the method for preparing validamycin is suitable for popularization and application in the industrial production of validamycin.
The products are all sold on the market, and the parts are parts by weight, and the concentration is mass concentration.
Detailed Description
The streptomyces strain of the present invention is a conventional streptomyces strain, and can be obtained commercially or isolated from rice by a method conventionally used by those skilled in the art. The recombinant strain can also be obtained by a method disclosed in literature validamycin strain natural breeding and prolificacy research (Nature science and sciences journal of the university of Hunan province 2009,32(2) Author Shihua, Qifengpei, Van Guang and Xuancai (chemical and chemical industry institute of the university of Hunan province, China Changsha 410081)).
In the examples, other products are commercially available products, the detection means is high performance liquid chromatography, and the detection conditions are as follows: the flow rate is 1ml/min, the detection wavelength is 210nm, an Elite Hypersil ODS25um, 4.6mm × 250mm analytical column is used, the column temperature is controlled at 25 ℃, and the retention time is about 8 min.
Example 1
The embodiment specifically provides a method for preparing validamycin by fermenting streptomyces hygroscopicus validamycin A. Comprises the following steps:
step (1): inoculating the streptomyces hygroscopicus validus var validus into a slant strain culture medium, culturing until spores are mature, and preparing a seed solution; the strain culture medium comprises the following components: 30g/L corn flour, 22g/L soybean cake powder and 10g/L, NaCl2 g/2 g/L, KH yeast powder2PO40.8g/L、CaCO30.2g/L and the balance of water, and adjusting the pH value to 7.0; standby;
step (2): inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid; the fermentation medium comprises the following components: 100g/L corn flour, 25g/L soybean cake powder, 5g/L, NaCl1g/L, KH g yeast powder2PO41.5g/L，CaCO30.5g/L of cellulase, 0.003g/L of cellulase and the balance of water; adjusting the pH value to 7.0, wherein the fermentation culture is shaking culture, the rotation speed of the shaking culture is 220r/min, the fermentation temperature is 37 ℃, and the fermentation time is 96 h;
and (3): extracting validamycin from the fermentation liquor.
The validamycin prepared in the embodiment is detected to have the content of 27000 mu g/mL.
Example 2
The embodiment specifically provides a method for preparing validamycin by fermenting streptomyces hygroscopicus validamycin A. Comprises the following steps:
step (1): inoculating the streptomyces hygroscopicus validus var validus into a slant strain culture medium, culturing until spores are mature, and preparing a seed solution; the strain culture medium comprises the following components: 25g/L corn flour, 20g/L soybean cake powder and 15g/L, NaCl1g/L, KH yeast powder2PO40.5g/L、CaCO30.1g/L and the balance of water, and adjusting the pH value to 7.2; standby;
step (2): inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid; the fermentation medium comprises the following components: 90g/L corn flour, 30g/L soybean cake powder and 8g/L, NaCl2g/L, KH g yeast powder2PO43g/L，CaCO30.6g/L, 0.001g/L of cellulase and the balance of water; adjusting the pH value to 7.2, wherein the fermentation culture is shaking culture, the rotation speed of the shaking culture is 220r/min, the fermentation temperature is 35 ℃, and the fermentation time is 90 hours;
and (3): extracting validamycin from the fermentation liquor.
The validamycin prepared in the embodiment is detected to have the content of 26800 mug/mL.
Example 3
The embodiment specifically provides a method for preparing validamycin by fermenting streptomyces hygroscopicus validamycin A. Comprises the following steps:
step (1): inoculating the streptomyces hygroscopicus validus var validus into a slant strain culture medium, culturing until spores are mature, and preparing a seed solution; the strain culture medium comprises the following components: 28g/L corn flour, 22g/L soybean cake powder and 13g/L, NaCl2 g/2 g/L, KH yeast powder2PO40.6g/L、CaCO30.15g/L and the balance of water, and adjusting the pH value to 6.8; standby;
step (2): inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain the hairFermenting liquid; the fermentation medium comprises the following components: 95g/L corn flour, 28g/L soybean cake powder and 7g/L, NaCl2g/L, KH g yeast powder2PO42g/L，CaCO30.5g/L, 0.002g/L of cellulase and the balance of water; adjusting the pH value to 7.5, wherein the fermentation culture is shaking culture, the rotation speed of the shaking culture is 250r/min, the fermentation temperature is 40 ℃, and the fermentation time is 110 h;
and (3): extracting validamycin from the fermentation liquor.
The validamycin prepared in the embodiment is detected to have the content of 26750 mug/mL.
Example 4
A fermentation medium comprising the following components: 90g/L corn flour, 25g/L soybean cake powder and 5g/L, NaCl1g/L, KH yeast powder2PO41g/L、CaCO30.4g/L, cellulase 0.001g/L and the balance of water.
Example 5
A fermentation medium comprising the following components: 100g/L corn flour, 30g/L soybean cake powder, 8g/L, NaCl2g/L, KH g yeast powder2PO43g/L、CaCO30.6g/L, cellulase 0.005g/L and the balance of water.
Comparative example 1
The only difference from example 1 is that the cellulase amount in step (2) of this example was controlled to have a concentration of 0.1g/L in the fermentation medium, and the validamycin prepared in this example was detected to have a content of 22000. mu.g/mL.
Comparative example 2
Compared with example 1, the difference is that the step (2) of this example does not contain the fibromycin, and the validamycin prepared in this example is detected to have a content of 21000. mu.g/mL.
Experiments show that: the addition of the cellulase in an amount of 0.001-0.005 g/L in the fermentation medium can obviously improve the yield of validamycin production.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for preparing validamycin from streptomyces hygroscopicus validamycin A, comprising the following steps:
step (1): inoculating the streptomyces hygroscopicus validus var validus into a slant strain culture medium, culturing until spores are mature, and preparing a seed solution for later use;
step (2): inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid;
the fermentation medium comprises the following components: 0.001-0.005 g/L cellulase, 90-100 g/L corn flour, 25-30 g/L soybean cake powder, 5-8 g/L, NaCl 1-1 g/2 g/L, KH yeast powder2PO41～3g/L、CaCO30.4-0.6 g/L and water, and adjusting the pH value to 7.0-7.5;
and (3): extracting validamycin from the fermentation liquor.
2. The method of claim 1, wherein the strain medium of step (1) comprises the following components: 25-30 g/L of corn flour, 20-22 g/L of soybean cake powder and 10-15 g/L, NaCl 1-3 g/L, KH of yeast powder2PO40.5～1g/L、CaCO30.1-0.2 g/L, and the balance of water; adjusting the pH value to 6.8-7.2.
3. The method of claim 2, wherein the strain medium of step (1) comprises the following components: 30g/L corn flour, 22g/L soybean cake powder and 10g/L, NaCl2g/L, KH g/yeast powder2PO40.8g/L、CaCO30.2g/L and the balance of water.
4. The method according to any one of claims 1 to 3, wherein in step (2), the pH of the fermentation medium is adjusted to 7.2.
5. Such as rightThe method according to any one of claims 1 to 3, wherein the fermentation medium in the step (2) comprises the following components: 0.001-0.005 g/L cellulase, 100g/L corn flour, 25g/L soybean cake powder and 5g/L, NaCl1g/L, KH yeast powder2PO41.5g/L、CaCO30.5g/L and water.
6. The method of any one of claims 1 to 3, wherein the water is distilled or deionized water.
7. The method according to claim 1, wherein the fermentation culture in the step (2) is shaking culture, the rotation speed of the shaking culture is 200-250 r/min, the fermentation temperature is 35-45 ℃, and the fermentation time is 90-110 h.
8. The method of claim 1, comprising the steps of:
step (1): inoculating the streptomyces hygroscopicus validus var validus into a slant strain culture medium, culturing until spores are mature, and preparing a seed solution; the strain culture medium comprises the following components: 30g/L corn flour, 22g/L soybean cake powder and 10g/L, NaCl2g/L, KH g/yeast powder2PO40.8g/L、CaCO30.2g/L and the balance of water, and adjusting the pH value to 7.0 for later use;
step (2): inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid; the fermentation medium comprises the following components: 100g/L corn flour, 25g/L soybean cake powder, 5g/L, NaCl1g/L, KH g yeast powder2PO41.5g/L、CaCO30.5g/L of cellulase, 0.003g/L of cellulase and the balance of water; adjusting the pH to 7.2;
and (3): extracting validamycin from the fermentation liquor.
9. A fermentation medium is characterized by comprising the following components: 90-100 g/L corn flour, 25-30 g/L soybean cake powder and 5-8 g/L, NaCl 1-2 g/L, KH yeast powder2PO41～3g/L、CaCO30.4-0.6 g/L, 0.001-0.005 g/L cellulase, and the balance water.
10. Use of the fermentation medium of claim 9 for the production of validamycin.
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| 井冈霉素产生菌的诱变选育及发酵条件优化;廖晓珣;《中国优秀硕士学位论文全文数据库 农业科技辑》;20080515(第5期);第22页2.5节以及第26页3.5.1节 * |
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